Involvement of Lipocalin-like CghA in Decalin-Forming Stereoselective Intramolecular [4+2] Cycloaddition

Abstract

Understanding enzymatic Diels-Alder (DA) reactions that can form complex natural product scaffolds is of considerable interest. Sch 210972 1, a potential anti-HIV fungal natural product, contains a decalin core that is proposed to form through a DA reaction. We identified the gene cluster responsible for the biosynthesis of 1 and heterologously reconstituted the biosynthetic pathway in Aspergillus nidulans to characterize the enzymes involved. Most notably, deletion of cghA resulted in a loss of stereoselective decalin core formation, yielding both an endo (1) and a diastereomeric exo adduct of the proposed DA reaction. Complementation with cghA restored the sole formation of 1. Density functional theory computation of the proposed DA reaction provided a plausible explanation of the observed pattern of product formation. Based on our study, we propose that lipocalin-like CghA is responsible for the stereoselective intramolecular [4+2] cycloaddition that forms the decalin core of 1.

Keywords: cycloaddition; decalin; density functional calculations; fungal metabolite; tetramic acid.

Figure 1

Proposed biosynthetic pathway of 1. (A) Formation of an unusual amino acid, γ-hydroxymethyl-l-glutamic acid 5, from two molecules of pyruvic acid dimerized by an aldolase CghB. (B) CghG-catalyzed synthesis of the linear substrate and a tetramic acid moiety, and the two final products that can be formed by the proposed Diels–Alder reaction. Abbreviations: KS, ketosynthase; MAT, malonyl-CoA acyltransferase; DH, dehydratase; MT, methyltransferase; KR, ketoreductase; ACP, acyl carrier protein; C, condensation; A, adenylation; T, thiolation; R, reductase; ER, enoyl reductase; SAM, S-adenosyl-l-methionine.

Figure 2

Confirmation of the activity of CghB by bioconversion of synthetic substrate 5 to 1 in C. globosum. HPLC traces of extracts of the culture of (i) wild-type control CGKW14, (ii) negative control of ΔcghB/CGKW14 without fed 5 and (iii) ΔcghB/CGKW14 with fed 5.

Figure 3

HPLC analysis of the metabolites from cghA strain of C. globosum. HPLC traces of extracts of the culture of (i) wild-type control CGKW14 and (ii) deletion mutant ΔcghA/CGKW14. The yield of 1 and 7 from the ΔcghA/CGKW14 culture were 2 and 1 mg/L, respectively, whereas 60 mg/L of 1 was isolated from the wild-type reference CGKW14 culture.

Figure 4

Confirmation of the role of CghA in the selective formation of 1 by complementing the cghA-disruption mutant with cghA in C. globosum. HPLC traces of extracts of the culture of (i) wild-type control CGKW14, (ii) ΔcazM/CGKW14 as a control showing the loss of production of 8 and 9, (iii) ΔcghA/ ΔcazM/CGKW14 and (iv) ΔcghA/cazM∷cghA/CGKW14. Authentic reference of (v) 1 and (vi) 7.

Figure 5

Reconstitution of the biosynthesis of 1 in Aspergillus nidulans A1145 and loss of stereoselective product formation upon elimination of cghA. HPLC traces of metabolic extracts from the culture of (i) wild-type A. nidulans A1145 control, (ii) A. nidulans harboring cghA, cghB, cghC and cghG and (iii) A. nidulans harboring cghB, cghC and cghG. Authentic reference of (iv) 1 and (v) 7.

Figure 6

Computational studies of the proposed Diels–Alder reaction involved in the biosynthesis of Sch 210972. (A) Truncated substrates examined using density functional theory. (B) Computed transition structures of the intramolecular Diels–Alder reaction of 10. The activation free energy (bold) and th e forming bond lengths are given. Unfavorable 1,3-diaxial interactions are indicated by opposing arcs.

Scheme 1

Natural products putatively biosynthesized via Diels–Alder reactions. Resulting cyclohexyl groups are shown in gray.

Scheme 2

The endo and exo transition state of the [4+2] cycloaddition, leading to the formation of 1 and 7, respectively.